Recombination between irradiated shuttle vector DNA and chromosomal DNA in African green monkey kidney cells.
An autonomously replicating shuttle vector was used to investigate enhancement of plasmid-chromosome recombination in mammalian host cells by gamma irradiation and UV light. Sequences homologous to the shuttle vector were stably inserted into the genome of African green monkey kidney cells to act as the target substrate for these recombination events. The shuttle vector molecules were irradiated at various doses before transfection into the mammalian host cells that contained the stable insertions. The homologous transfer of the bacterial ampicillin resistance gene from the inserted sequences to replace a mutant ampicillin sensitivity gene on the shuttle vector was identified by the recovery of ampicillin-resistant plasmids after Hirt extraction and transformation into Escherichia coli host cells. Gamma irradiation increased homologous shuttle vector-chromosome recombination, whereas UV light did not increase the frequency of recombinant plasmids detected. Introducing specific double-strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was increased by UV irradiation of the plasmid but did not change with time. The ampicillin-resistant recombinant plasmid molecules analyzed appeared to rise mostly from nonconservative exchanges that involved both homologous and possibly nonhomologous interactions with the host chromosome. The observation that similar recombinant structures were obtained from all the plasmid treatments and host cells used suggests a common mechanism for plasmid-chromosome recombination in these mammalian cells.
Mudgett
J S
JS
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Taylor
W D
WD
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002522:Chlorocebus aethiops; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D005720:Gamma Rays; D005822:Genetic Vectors; D009154:Mutation; D010957:Plasmids; D011995:Recombination, Genetic / Q000528:radiation effects*; D013997:Time Factors; D014162:Transfection; D014466:Ultraviolet Rays